All cells secrete proteins constitutively, but specialized polypeptide- secreting neuroendocrine cells have developed an additional pathway of storage of peptide hormones in granules whose exocytosis is acutely regulated by specific physiologic stimuli. Since storage granules are enriched in proteins different from those secreted constitutively and those concentrated in lysosomes, professional secretory cells must internally distinguish these different classes of proteins by a complex process known collectively as molecular sorting and targeting. Such a process has great biological significance, because this basic mechanism, in conjunction with stimulus-secretion coupling in endocrine cells, serves as a primary line of defense against disturbances in metabolic homeostasis. In an array of human/animal diseases, alterations in molecular sorting of the above classes of molecules are associated with abnormalities ranging from subtle phenotypic defects to lack of viability. This grant continues to focus on gaining a working knowledge of molecular mechanism's involved in protein sorting in the secretory pathway. Much progress has been made in the past 4 years; however, considerable controversy remains over two different models, which are not mutually exclusive, that have been created to explain how peptide hormones and other luminal proteins are delivered to, and stored within, secretory granules. The first of these two views is a "sorting for entry" model in which a subset of proteins are pre-selected in the trans-Golgi network for entry into storage granules while other proteins are efficiently excluded from entry. The second model is termed "sorting by retention" in which entry into storage granules does not require participation in an insoluble protein complex nor to a receptor in the forming granule membrane. In this view, entry into forming granules may not be exclusive to regulated secretory proteins; however, higher-order intermolecular interactions (condensation) within maturing granules facilitates protein retention within the granule compartment. At the same time, an exit route efficiently removes certain selected proteins from maturing granules by receptor-mediated vesicle budding, accompanied by the passive removal of a fraction of secretory proteins that have inefficient retention properties. The goals of this grant application continue to be to explore aspects of this sorting-by- retention hypothesis. Important preliminary findings are presented which establish the feasibility of novel experiments underlying 3 Specific Aims.